The overall goal of this proposal is to gain a better understanding of the central regulation of the hypothalamic- pituitary-thyroid axis (HPT axis) by thyroid hormones (T4 and T3) and thyrotropin-releasing hormone (TRH). Thyroid stimulating hormone (TSH also known as thyrotropin) is a heterodimeric protein, which is synthesized and secreted from the thyrotroph and is essential for activating thyroid hormone (TH) synthesis in the thyroid. THRB isoforms (principally the THRB2 isoform) mediates T3 negative feedback at the level of the hypothalamus and pituitary thyrotroph based on elevated TH and TSH levels in THRB isoform KO mice. In contrast, THRA1 KO mice actually display slightly reduced TH levels associated with slightly elevated TSH levels. These findings suggest that THRA1 has no role in negative T3 regulation of the HPT axis in the euthyroid state. However, THRA1 must play some role since KO of all THR isoforms (THRA1, THRB1 and THRB2) markedly elevates both TSH and TH levels above those observed in THRB KO mice. What explains this THR isoform difference? We hypothesize that THRB isoforms bind preferentially to a negative thyroid response element(s) (nTREs) in the Tshb gene to mediate negative T3 regulation. In the absence of T3, other workers have suggested that TRH and TSH production are maximal, and that the unliganded THRs play no direct role in activating the axis. Our in vitro studies and studies in KO mice challenge this model. We have demonstrated, for example, that the N-terminus of THRB2 contains a potent trans-activation domain and that both THRB2 and hypothalamic TRH are necessary for full activation of Tsh subunit gene expression in the hypothyroid state in vivo. We hypothesize that specific THR isoforms mediate an increase in histone acetylation on the Tshb gene, which underlies the mechanism by which the unliganded THRB2 activates basal gene expression. Finally, we have characterized a unique, permanent mouse thyrotroph cell line (TT1.1) that displays physiological T3 negative regulation of the Tshb gene. We find that acute changes in histone methylation of the proximal Tshb gene occur after T3 treatment. We hypothesize that liganded THRs recruit a histone demethylase to Tshb resulting in a decrease in histone methylation and T3- inhibition of gene expression.